Phenotypes of bd, bdf, and wild-type Dazao larvae.

The epidermis of bd is dark gray, and the epidermis of bdf is light gray at the 5th instar (day 3) of silkworm larvae. The bar indicates 1 centimeter.

Positional cloning of the bd locus.

(A) We used 532 BC1 individuals to map the bd locus between PCR markers c and i. The numbers above the DNA markers indicate recombination events.

(B) A total of 1162 BC1 individuals were used to narrow the bd locus to an approximately 400 kb genomic region.

(C) Partial enlarged view of the region responsible for bd. This region contains 4 predicted genes, BGIBMGA012517, BGIBMGA012518, BGIBMGA012519 and BGIBMGA014089.

(D) Analysis of nucleotide differences in the region responsible for bd. The green block indicates the deletion of the genome in bd mutants. The black vertical lines indicate the SNPs and indels of bdf mutants.

Spatiotemporal expression of Bm-mamo.

(A) Temporal expression of Bm-mamo. In the molting stage and adult stage, this gene was significantly upregulated. M: molting stage, W: wandering stage, P: pupal stage, P1: Day 1 of the pupal stage, A: adult stage, 4th3d indicates the 4th instar on Day 3. 1st to 5th denote the first instar of larvae to fifth instar of larvae, respectively.

(B) Tissue-specific expression of 4th-instar molting larvae. Bm-mamo had relatively high expression levels in the midgut, head, and epidermis. AMSG: anterior division of silk gland and middle silk gland, PSG: posterior silk gland.

(C) Detailed analysis of Bm-mamo at the 4th larval stage in the epidermis of the Dazao strain. Bm-mamo expression is upregulated during the molting stage. HCS indicates the head capsule stage. The “h” indicates the hour, 90 h: at 90 hours of the 4th instar.

(A) siRNA was introduced by microinjection followed by electroporation. “+” and “-” indicate the positive and negative poles of the electrical current, respectively. Scale bars, 1 cm.

(B) Partial magnification of the siRNA experimental group and negative control group. Scale bars, 0.2 cm.

(C) and (D) Relative expression levels of Bm-mamo in the negative control and RNAi groups were determined by qPCR analysis. The means ± s.d.s. Numbers of samples are shown in the upper right in each graph. **P<0.01, paired Student’s t test (NS, not significant).

(E) Statistical analysis of the efficiency of the RNAi.

Bm-mamo knockout

(A) Larval phenotype of G3 fourth-instar larvae of Dazao targeted for Bm-mamo. Scale bars, 1 cm.

(B) Partial magnification of the Bm-mamo knockout individual and control. Scale bars, 0.2 cm.

(C) After 48 hours of egg laying, the pigmentation of the eggs indicates that those produced by knockout homozygous females cannot undergo normal pigmentation and development.

(D) Genomic structure of Bm-mamo. The open reading frame (blue) and untranslated region (black) are shown. The gRNA 1 and gRNA 2 sequences are shown.

(E) Sequences of the Bm-mamo knockout individuals. Lines 1 and 2 indicate deletions of 15 and 71 bp, respectively.

Melanin metabolism pathway and qPCR of related genes.

(A) The melanin metabolism pathway. Blue indicates amino acids and catecholamines, red indicates the names of genes, and black indicates the names of enzymes.

(B) Relative expression levels of eight genes in the heterozygous Bm-mamo knockout group (blue) and homozygous Bm-mamo knockout group (red) determined via qPCR analysis. The means ± s.d.s. *P<0.05, paired Student’s t test. 4th3d indicates the 4th instar on Day 3, M indicates molting, and h indicates hours.

Heatmap of 28 differentially expressed cuticular protein genes.

Differentially expressed cuticular protein genes between homozygous (mamo-/mamo-) and heterozygous (mamo-/+) Bm-mamo knockout individuals. Red indicates upregulation in homozygous individuals. Blue indicates downregulation in homozygous individuals. White indicates no difference in expression level or no investigation. 4th1d indicates 4-instar Day 1, M indicates molting, and h indicates hours.